The present invention relates generally to the field of molecular biology. More particularly, it concerns the replication and packaging of recombinant adeno-associated viral-based vectors, and a scaleable process for their large-scale production.
1.2.1 Adeno-associated Virus
Adeno-associated virus-2 (AAV)-2 is a human parvovirus that can be propagated both as a lytic virus and as a provirus (Cukor et al., 1984; Hoggan et al., 1972). The viral genome consists of linear single-stranded DNA (Rose et al., 1969), 4679 bases long (Srivastava et al., 1983), flanked by inverted terminal repeats of 145 bases (Lusby and Berns, 1982). For lytic growth AAV requires co-infection with a helper virus. Either adenovirus (Ad; Atchinson et al., 1965; Hoggan, 1965; Parks et al., 1967) or herpes simplex virus (HSV; Buller et al., 1981) can supply the requisite helper functions. Without helper, there is no evidence of AAV-specific replication or gene expression (Rose and Koczot, 1972; Carter et al., 1983). When no helper is available, AAV persists as an integrated provirus (Hoggan, 1965; Berns et al., 1975; Handa et al., 1977; Cheung et al., 1980; Berns et al., 1982).
Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact in the provirus. The ability of AAV to integrate into host DNA is apparently an inherent strategy for insuring the survival of AAV sequences in the absence of the helper virus. When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (Hoggan, 1965).
AAV sequences cloned into prokaryotic plasmids are infectious (Samulski et al., 1982). For example, when the wild type AAV/pBR322 plasmid, pSM620, is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues (Samulski et al., 1982). This renders it possible to modify the AAV sequences in the recombinant plasmid and, then, to grow a viral stock of the mutant by transfecting the plasmid into human cells (Samulski et al., 1983; Hermonat and Muzyczka, 1984).
AAV contains at least three phenotypically distinct regions (Hermonat and Muzyczka, 1984). The rep region codes for one or more proteins that are required for DNA replication and for rescue from the recombinant plasmid, while the cap and lip regions appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (Hermonat and Muzyczka, 1984). It has been shown that the AAV termini are required for DNA replication (Samulski et al., 1983).
The construction of two E. coli hybrid plasmids, each of which contains the entire DNA genome of AAV, and the transfection of the recombinant DNAs into human cell lines in the presence of helper adenovirus to successfully rescue and replicate the AAV genome has been described (Laughlin et al., 1983; Tratschin et al., 1984a; 1984b).
1.2.2 rAAV Vectors as Vehicles for Gene Therapy
Recombinant adeno-associated virus (rAAV) vectors have important utility as vehicles for the in vivo delivery of polynucleotides to target host cells (Kessler et al., 1996; Koeberl et al., 1997; Kotin, 1994; Xiao et al., 1996). rAAV vectors are useful vector for efficient and long-term gene transfer in a variety of mammalian tissues, e.g., lung (Flotte, 1993), muscle (Kessler, 1996; Xiao et al., 1996; Clark et al., 1997; Fisher et al., 1997), brain (Kaplitt, 1994; Klein, 1998) retina (Flannery, 1997; Lewin et al., 1998), and liver (Snyder, 1997).
It has also been shown that rAAV can evade the immune response of the host by failing to transduce dendritic cells (Jooss et al., 1998). Clinical trials have been initiated for several important mammalian diseases including hemophilia B, muscular dystrophy and cystic fibrosis (Flotte et al., 1996; Wagner et al., 1998).
1.2.3 Contemporary Methods for Preparing rAAV Vectors
Currently, rAAV is most often produced by co-transfection of rAAV vector plasmid and wt AAV helper plasmid into Ad-infected 293 cells (Hermonat and Muzyczka, 1984). Recent improvements in AAV helper design (Li et al., 1997) as well as construction of non-infectious mini-Ad plasmid helper (Grimm et al., 1998; Xiao et al., 1998; Salvetti, 1998) have eliminated the need for Ad infection, and made it possible to increase the yield of rAAV up to 105 particles per transfected cell in a crude lysate. Scalable methods of rAAV production that do not rely on DNA transfection have also been developed (Chiorini et al., 1995; Inoue and Russell, 1998; Clark et al., 1995). These methods, which generally involve the construction of producer cell lines and helper virus infection, are suitable for high-volume production.
The conventional protocol for downstream purification of rAAV involves the stepwise precipitation of rAAV using ammonium sulfate, followed by two or preferably, three rounds of CsCl density gradient centrifugation. Each round of CsCl centrifugation involves fractionation of the gradient and probing fractions for rAAV by dot-blot hybridization or by PCR(trademark) analysis.
A major problem associated with the use of rAAV vectors has been the difficulty in producing large quantities of high-titer vector stocks (Clark et al., 1995, Clark et al., 1996). The standard production protocol involves low-efficiency transfection of plasmid DNA containing the rep and cap genes and a plasmid containing the rAAV provirus with inverted terminal repeats. Cells are then superinfected with adenovirus to provide essential helper functions required for rAAV production.
Alternative procedures have been developed to improve the efficiency of rAAV production by delivering rep, cap and the adenovirus helper genes. These technologies have included the generation of rep and cap inducible cell lines and plasmids expressing the essential adenovis helper genes (Clark et al., 1995; Clark et al., 1996; Vincent et al., 1990; Xiao et al., 1998; Grimm et al., 1998). Although these techniques have improved the yield of rAAV production, they have not been entirely satisfactory. Procedures employing transfection methods are not efficient, and tend to be extremely variable in yield from preparation to preparation. Moreover, such procedures are difficult to scale up to produce the large quantity of rAAV vector needed for clinical trials.
The production of rep and cap inducible cell lines is a particular challenge because the yield of rAAV produced from different clones is variable and does not exceed the efficiency of transfection methods (Clark et al., 1995; Clark et al., 1996, Vincent et al., 1990). Production procedures for rAAV that utilize adenovirus and transfection of rep and cap containing plasmids have the potential to generate wild type AAV (wt AAV) through illegitimate recombination of the ITRs with rep and cap sequences. This leads to preferential amplification of the wt AAV genome over the rAAV genome.
A major drawback in the use of rAAV vectors for gene transfer studies in vivo and their application to clinical procedures, such as that of gene therapy, has been the difficulty in producing large quantities of rAAV vector. For the therapeutic correction of some diseases, it is estimated that 1xc3x971014 rAAV particles must be administered per patient. This will require the culture of greater than 1xc3x971012 cells to produce the quantity of rAAV vector that will be needed to therapeutically treat each patient. The use of contemporary transfection methods on this scale of rAAV production is extremely problematic, costly and time consuming.
The development of a packaging system that provides all the helper functions needed for rAAV production from a rAAV producer cell line would greatly facilitate the large-scale production of rAAV. Transfection procedures would not be required and the producer cell line could be grown in large quantities at high densities in commercially available laboratory equipment.
The present invention overcomes these and other inherent limitations in the prior art by providing packaging systems that provide all of the required helper functions, and methods for the large-scale production of rAAV. The present invention demonstrates the ability of a recombinant herpes simplex virus (rHSV) or a rHSV amplicon expressing AAV Rep and Cap to support replication and packaging of rAAV. The present methods overcome the need for transfection procedures, and provide reliable, cost-effective means for generating large quantities of rAAV. Superinfection of appropriate host cell cultures with the vectors described herein produces quantities of rAAV not attainable by any other means. By providing a second virus or cell line that contains the rAAV provirus, the present methods overcome the significant problem of spontaneous deletions in the AAV ITR when growing rAAV-based plasmids in bacterial cell cultures.
The present invention provides the first system that supplies AAV genes rep and cap and the HSV-1 helper functions needed for rAAV production in one delivery vehicle. The rHSV-1 and rHSV-1 amplicon-based vector systems supply Rep, Cap and the HSV-1 helper functions required for rAAV production. Amplicon and virus stocks have been produced that express Rep and Cap from their native promoters (p5, p19 and p40). To increase the yield of rAAV production and make the rHSV-1 and rHSV-1 amplicon systems practical alternatives to adenoviral systems for rAAV production, HSV-1 amplicon and vector systems that expresses Rep and Cap from their native promoters and uses an ICP27 mutated HSV-1 virus, d27-1, as the genetic background of the amplicon or vector has been developed. Use of the defective HSV-1 amplicon or vector results in rAAV production with an efficiency that exceeds previously described methods (Flotte et al., 1995). Southern blot and PCR(trademark) analyses have shown that no wt AAV were produced using these modified amplicons or helper viruses. The present system provides means for increasing the scale of rAAV production to a level such that sufficient rAAV can now be produced for preclinical and clinical trials utilizing rAAV-based vectors for gene delivery.
The present invention provides DNA segments comprising an AAV rep coding sequence operably linked to a promoter, an AAV cap coding sequence operably linked to a promoter, an HSV-1 origin of replication and an HSV-1 packaging sequence. In preferred embodiments, the AAV rep coding sequence and/or the AAV cap coding sequence is operably linked to a p5, p19 or p40 promoter. In certain embodiments, the DNA segment is comprised within a recombinant herpes simplex virus vector, or within a recombinant herpes simplex virus capsid.
As used herein in this context, the term xe2x80x9crecombinant herpes simplex virus vectorxe2x80x9d will be understood to mean genomic DNA of the herpes simplex virus with non-herpes simplex virus DNA added by the hand of man. The term xe2x80x9crecombinant herpes simplex virus capsidxe2x80x9d, as used herein in this context will be understood to mean the herpes simplex virus head, comprised of herpes simplex virus capsid proteins, comprising a recombinant DNA segment, such as a plasmid, cosmid or the in like, that comprises at least an HSV-1 origin of replication and an HSV-1 packaging sequence.
Thus, the present invention also provides recombinant herpes simplex virus vectors comprising an AAV rep coding sequence operably linked to a promoter and an AAV cap coding sequence operably linked to a promoter. In preferred aspects of the invention, the AAV rep coding sequence and/or the AAV cap coding sequence is operably linked to a p5, p19 or p40 promoter.
In certain recombinant herpes simplex virus vectors of the present invention, a non-essential HSV gene is altered. In particular embodiments, the non-essential HSV gene is altered to increase expression. In a general sense, genes that encode proteins that are beneficial to the host cell, or that increase the production of rAAV particles are contemplated for such alteration. Examples of non-essential HSV genes that are altered to increase expression includes, but is not limited to, the HSV gene encoding ICP8.
In other embodiments, the non-essential HSV gene is mutated, such as by one or more point mutants or insertions, or substantially or completely deleted, such that the gene product of the non-essential HSV gene is either non-functional or absent. In a general sense, genes that encode proteins that are deleterious to the host cell, or that decrease the production of rAAV particles are contemplated for such alteration. Examples of non-essential HSV genes that are contemplated for mutation or deletion include, but are not limited to, the HSV genes encodes ICP27, an HSV late gene and/or glycoprotein H.
In preferred embodiments of the invention, the recombinant vector is comprised within a recombinant herpes simplex virus. As used herein in his context, the term xe2x80x9crecombinant herpes simplex virusxe2x80x9d will be understood to mean a complete herpes simplex virus that comprises a xe2x80x9crecombinant herpes simplex virus vectorxe2x80x9d, as defined above.
Therefore, the present invention further provides recombinant herpes simplex viruses comprising an AAV rep coding sequence operably linked to a promoter and an AAV cap coding sequence operably linked to a promoter. In preferred aspects of the invention, the AAV rep coding sequence and/or the AAV cap coding sequence is operably linked to a p5, p19 or p40 promoter.
In certain recombinant viruses of the present invention, a non-essential HSV gene is altered. In particular embodiments, the non-essential HSV gene is altered to increase expression. Examples of non-essential HSV genes that are altered to increase expression includes, but is not limited to, the HSV gene encoding ICP8. In other embodiments, the non-essential HSV gene is mutated, such as by one or more point mutants or insertions, or substantially or completely deleted, such that the gene product of the non-essential HSV gene is either non-functional or absent. Examples of non-essential HSV genes that are contemplated for mutation or deletion include, but are not limited to, the HSV genes encodes ICP27, an HSV late gene and/or glycoprotein H. In preferred embodiments, the recombinant virus is the d27.1rc virus.
The present invention also provides kits comprising, in a suitable container, a DNA segment comprising an AAV rep coding sequence operably linked to a promoter, an AAV cap coding sequence operably linked to a promoter, an HSV-1 origin of replication and an HSV-1 packaging sequence. In further aspects of the invention, the kit comprises an HSV-1 helper virus. In preferred aspects, a non-essential gene of the HSV-1 helper virus is altered. As detailed above, in certain aspects of the invention, a non-essential gene of the HSV-1 helper virus, exemplified by, but not limited to the gene encoding ICP8, is altered to increase expression. In other aspects, a non-essential gene of the HSV-1 helper virus, including, but not limited to the genes encoding ICP27 and/or glycoprotein H, is mutated or substantially deleted In certain preferred embodiments, the HSV-1 helper virus is the d27.1 HSV-1 virus.
Additionally, the present invention provides kits comprising, in a suitable container, a recombinant herpes simplex virus vector comprising an AAV rep coding sequence operably linked to a promoter and an AAV cap coding sequence operably linked to a promoter. In preferred kits of the invention, the recombinant herpes simplex virus vector is comprised in a recombinant herpes simplex virus.
The present invention also provides methods for preparing a rAAV comprising providing an HSV-1 helper virus and a DNA segment comprising an AAV rep coding sequence operably linked to a promoter, an AAV cap coding sequence operably linked to a promoter, an HSV-1 origin of replication and an HSV-1 packaging sequence to a host cell that comprises a rAAV, culturing the cell under conditions effective to produce rAAV in the cell, and obtaining the rAAV from the cell. As used herein in this context, the term xe2x80x9chost cell that comprises a rAAVxe2x80x9d will be understood to include a host cell that comprises a rAAV provirus integrated into the genome of the host cell, as well as a host cell that is infected with a rAAV. Thus, in certain aspects, the host cell comprises the rAAV integrated into the genome of the cell, while in other aspects the host cell is provided with the rAAV, the HSV-1 helper virus and the DNA segment simultaneously.
Preferred host cells include, but are not limited to, HeLa, 293 or Vero cells. In certain preferred methods of the invention, the rAAV comprises an AAV-2 genome. However, while the preferred rAAV genome is generally the AAV-2 genome, the capsid can be from any serotype of AAV. Therefore, in particular methods, the rAAV comprises an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5 or AAV-6 capsid. As the present compositions and methods are designed for large-scale production of rAAV vectors, in preferred embodiments, the rAAV comprises a therapeutic gene. In certain methods, the AAV rep coding sequence and/or the AAV cap coding sequence is operably linked to a p5, p19 or p40 promoter. In other methods, at least a first AAV capsid protein is operably linked to an HSV late promoter, such as the HSV 110 promoter.
As detailed above, in certain methods of the present invention a non-essential gene of the HSV-1 helper virus is altered. In certain methods, a non-essential gene of the HSV-1 helper virus, exemplified by, but not limited to the gene encoding ICP8, is altered to increase expression. In other methods, a non-essential gene of the HSV-1 helper virus, including, but not limited to the genes encoding ICP27 and/or glycoprotein H, is mutated or substantially deleted. In certain preferred methods, the HSV-1 helper virus is the d27.1 HSV-1 virus. Thus, the present invention further provides a recombinant AAV virus produced by any of the methods of the present invention, as well as kits comprising, in a suitable container, a recombinant AAV virus produced by any of the methods of the present invention.
The present invention additionally provides methods for preparing a rAAV comprising providing a recombinant herpes simplex virus that comprises an AAV rep coding sequence operably linked to a promoter and an AAV cap coding sequence operably linked to a promoter to a host cell that comprises a rAAV, culturing the cell under conditions effective to produce rAAV in the cell, and obtaining the rAAV from the cell.
As detailed above, in certain methods a non essential gene of the recombinant herpes simplex virus, such as the gene encoding ICP8, is altered to increase expression, while in other methods, a non-essential gene of the recombinant herpes simplex virus, such as the gene encoding ICP27 or glycoprotein H, is mutated or substantially or completely deleted.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows a map of pHSV-RC, which was used to generate amplicons that replicate and package rAAV virions. The plasmid is a pUC-based vector. The a-sequence contains the HSV-1 packaging signals and is cloned into the EcoRI site. The 110-sequence contains an HSV-1 origin of replication and is the internal SmaI fragment from the HSV-1 ori S. The 110-sequence is inserted in the SmaI site. (The 110 and a-sequence containing plasmid is p110a) Rep and cap are the AAV-2 rep and cap genes isolated from psub201 by an XbaI digest and cloned into the XbaI site of p110a to create pHSV-RC.
FIG. 2 shows the integration vector used to produce d27.1-rc. The plasmid pHSV-106 contains the BamHI fragment encoding the tk gene of HSV-1. The AAV-2 rep and cap genes, under control of their native promoters, were cloned into the KpnI site of tk gene to generate pHSV-106-rc. Restriction digest of pHSV-106-rc with SphI was used to generate the linear fragment. This fragment was cotransfected with d27.1-lacZ infected cell DNA into V27 cells to generate d27.1-rc by homologous recombination.
FIG. 3 demonstrates that recombinant adeno-associated virus can be amplified after coinfection with d27.1-rc. 293 cells were transfected with AAV-GFP proviral plasmid. Approximately 3xc3x97107 cells were present in each group. 24 h after transfection, the cells were superinfected with different MOIs of d27.1-rc. 36 h post infection, a cell lysate was made from the infected cells by three rounds of freeze-thaw. The viral lysate was heat inactivated at 55xc2x0 C. for one hour and then titered in duplicate on C12 cells that were coinfected with Ad (MOI of 20). 48 h post infection the C12 cells were analyzed for GFP expression using fluorescent microscopy and a titer was determined (in expression units). The amount of AAV-GFP produced per transfected cell was then calculated. This study was repeated three times.
FIG. 4 illustrates that the vector d27.1-rc can produce rAAV from a proviral cell line. The cell line GFP-92 is a 293 derived cell line that has a single copy of AAV-GFP integrated into its genome. The vector d27.1-rc was used to produce AAV-GFP from this cell line. 1.5xc3x97107 GFP-92 cells were infected with d27.1-rc at different MOIs. 48 h post-infection a cell lysate was made from the infected cells by three rounds of freeze-thaw. The viral lysate was heat inactivated at 55xc2x0 C. for one hour and then titered in duplicate on C12 cells that were coinfected with Ad (MOI of 20). 48 h post-infection the C12 cells were analyzed for GFP expression using fluorescent microscopy and a titer was determined (expression units). The amount of AAV-GFP produced per transfected cell was then calculated. This study was repeated three times.